Identification of apple cultivars hypoallergenic for birch pollen‐allergic individuals by a multidisciplinary in vitro and in vivo approach

Abstract Background Birch pollen‐related apple allergy is the most frequent IgE‐mediated food allergy in Central‐Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year‐round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long‐term storage and by clinical challenges of allergic individuals before and after the birch pollen season. Methods Proteins were extracted from fruits of ‘Santana’, ‘Golden Delicious’ (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1‐levels were analysed by mass spectrometry, SDS‐PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty‐eight allergic individuals underwent single‐blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen‐specific IgE‐levels were determined. Results After storage all cultivars except ‘Santana’ were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen‐specific IgE‐levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. Conclusions Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long‐term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

K E Y W O R D S
apple, basophil activation test, birch pollen-associated food allergy, Mal d 1

| INTRODUCTION
Birch pollen-related food allergy is the most common IgE-mediated food allergy in Central and Northern Europe and often caused by apple fruits (Malus domestica). [1][2][3][4] Birch pollen-related apple allergy (BPRAA) is predominantly caused by the major apple allergen, Mal d 1, which is structurally related to the major birch pollen allergen, Bet v 1. 2,[5][6][7][8] Following respiratory sensitization, a fraction of Bet v 1specific IgE antibodies cross-reacts with Mal d 1 and may cause immediate reactions to fresh apples. Most commonly this crossreactivity manifests as the so-called oral allergy syndrome (OAS) which is characterized by itching, tingling, and swelling in the mouth or oral angioedema. 9 Moreover, Mal d 1 may activate Bet v 1-specific effector T cells and trigger allergic late phase reactions which appear as an aggravation of atopic eczema. 4,[10][11][12][13] In many individuals BPRAA persists perennially and worsens during or shortly after the birch pollen season. 2,14 Consequently, around 9% of individuals living in Central Europe refrain from consuming fresh apples. 15 However, these fruits represent an important domestic source of vitamins, secondary plant metabolites such as phenolic compounds, carbohydrates, and fibers in the diet of the population in areas where birch trees are abundant. Therefore, "hypoallergenic" apple cultivars tolerable by birch pollen-allergic individuals will help to comply with their nutritional needs.
In the past, numerous studies have compared the allergenic potential of different cultivars by oral challenges and/or skin testing of individuals with BPRAA. [16][17][18][19][20][21][22][23][24] Concordantly, 'Golden Delicious' (GD) was defined as a cultivar of high and 'Santana' as a cultivar of low allergenic potential. 17,[25][26][27] Due to positive agronomic and economic traits, good taste, and year-round availability, GD is the most popular Malus domestica cultivar in Europe for decades. In contrast, the fruits of Santana cannot be stored longer than 3 months after harvest and become soft and inedible thereafter. Even modern storage conditions, such as controlled atmosphere storage and treatment with 1methylcyclopropene (1-MCP), hardly overcome this disadvantage.
Several other hypoallergenic apple varieties described so far also have significant disadvantages regarding cultivation and storability.
Consequently, the number of hypoallergenic cultivars with good agronomic performance (also for organic farming), excellent storability and quality of the fruits is still small.
A multidisciplinary approach was set to characterize the allergenicity of three very well storable genuine cultivars with suspected low allergenicity for individuals with BPRAA. The identification and quantification of Mal d 1 by mass spectrometry and immunological assays were complemented with basophil activation tests (BAT) and clinical challenges of allergic individuals. Furthermore, storage conditions known to enhance the concentration of Mal d 1 28-31 as well as pollen exposure known to increase the clinical reactivity of allergic individuals were considered. 2,14 Figure 1 summarizes the longitudinal study protocol.

| SDS-PAGE AND IMMUNOBLOTS
Aliquots of all protein extracts were thawed once and the protein content was measured by BCA (Thermo Fisher Scientific). Proteins

| BASOPHIL ACTIVATION TESTS (BAT)
Heparinized blood from individuals with BPRAA was incubated with   35 Intraclass correlation coefficients were computed which illustrate the importance of the random intercept for participant. All tests were two-tailed and differences were considered significant if p ≤ 0.05.
Given the exploratory nature of the study we did not consider multiple testing correction for the different endpoints. performed. An anti-BIP2 mAb served as loading control ( Figure 2D).

| Mal d 1 measurements
These analyses unanimously revealed enhanced allergen concentrations after long-term storage in all cultivars and were confirmed by competitive ELISA (Table 1).

| DISCUSSION
Careful monitoring of Mal d 1 levels in the course of storage of four cultivars with an initial concentration of less than 2 μg allergen/g fresh weight has revealed highest concentrations after 28-36 weeks at 4°C without treatment with 1-MCP. 31, 37 We applied similar conditions to trigger an increase of allergen in the studied cultivars.
Untargeted proteomics confirmed a rise of Mal d 1 among all proteins. We also found Mal d 1.02 as the most abundant isoform in the fruits of 'Bay 4152', 'Bay 4210', and 'Santana', matching earlier observations from quantitative PCR. 29 As this isoform has been described as less allergenic than Mal d 1.01, 38 we performed competitive ELISA with recombinant Mal d 1.0108 known to induce dose-dependent skin reactions and OAS in individuals with BPRAA. [39][40][41] Hereby assessed levels in the fruits of 'Santana' were in the range reported by others. 16,24,31,42,43 The levels in the cultivars in  (Fig. S2). 18